Antimicrobial Activity of Liposomal and Non-Liposomal Vaginal Suppositories with Origanum Compactum Essential

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M. Ibišević, S. Pilipović, I. Nešić, V. Kerleta, D. Husejnagić, E. Cilović Kozarević, E. Horozić, E. Karić, “Antimicrobial...“, Technologica Acta, vol. 13, no. 2, pp. 5-10, 2020. 5

ANTIMICROBIAL ACTIVITY OF LIPOSOMAL AND NON-LIPOSOMAL VAGINAL SUPPOSITORIES

WITH ORIGANUM COMPACTUM ESSENTIAL OIL ORIGINAL SCIENTIFIC PAPER

Merima Ibišević1, Saša Pilipović2, Ivana Nešić3, Vera Kerleta1, Darja Husejnagić4, Ermina Cilović Kozarević1, 5 1 Emir Horozić , Enida Karić DOI: 10.5281/zenodo.4540051

RECEIVED ACCEPTED 1 Faculty of Pharmacy, University of Tuzla, Tuzla, Bosnia and Herzegovina 2020-05-26 2020-07-21 2 The Agency for medicinal products and medical devices, Control laboratory of the Agency in Sarajevo, Bosnia and Herzegovina 3 Department of Pharmacy, University of Niš, Faculty of Medicine, Niš, Serbia 4 Faculty of Natural Sciences and Mathematics, University of Tuzla, Tuzla, BiH 5 Faculty of Technology, University of Tuzla, Tuzla, BiH

 [email protected]

ABSTRACT: Origanum compactum (Lamiaceae) is an endemic species of oregano from Morocco, and the main components are carvacol and thymol, which are considered to have antimicrobial activity. Essential oils can be unstable, poorly soluble in water and poorly delivered to target cells. The incorporation of essential oils into liposomes can reduce their irritant effect, while at the same time prolonging the action of the preparation itself as well as increasing its effectiveness. The aim of our study was to investigate antimicrobial activity of liposomal and non-liposomal vaginal suppositories, and see if there are any differences in antimicrobial activity. Examination of the antimicrobial activity of vaginal suppositories was examined in the same way as the antimicrobial activity of the essential oil, by the disk diffusion method. There were used standard bacterial strains from ATCC collection: Staphylococcus aureus (S. aureus) ATCC 25923, Enterococcus faecalis (E. faecalis) ATCC 51299, Escherichia coli (E. coli) ATCC 25922, Candida albicans (C. albicans) ATCC 10231. Liposomal vaginal suppositories had a smaller inhibition zones probably due to the slower release of active components, but still have an advantage over non- liposomal vaginal suppositories because they reduce the irritating potential of the essential oil.

KEYWORDS: vaginal suppository, liposome, essential oil, Origanum compactum, antimicrobial activity

INTRODUCTION are most often used as a bases. The carrier may, if necessary, contain emulsifiers and other excipients as Vaginitis is one of the most common infectious well as preservatives. Suppositories and vaginal diseases of the female genital system. The most suppositories are made in the main practice by the common causes of vaginitis are candidiasis and method of melting and pouring into molds. This bacterial vaginosis [1]. The incorporation of method implies that the drug substances are dissolved antimicrobial drugs into liposomes (and other or uniformly suspended or emulsified in a dissolved nanosystems) could significantly improve local base and the mixture is poured into appropriate vaginal therapy for fungal and bacterial infections. In molds. The most commonly used bases are different this way, it would be possible to use lower doses of types of Witepsol, which belong to the semi-synthetic the drug than with conventional (classic) forms of lipophilic base. They have advantages over cocoa drugs [2]. The vaginal cavity is an important area of butter because they have a defined melting point, and the reproductive tract and acts as a favorable site for are chemically more stable. drug administration due to avoiding the first-pass Origanum compactum Benth. (Lamiaceae) is an effect, large permeable area, rich vascularization, and endemic species of oregano from Morocco, and the relatively low enzyme activity [3]. main components are carvacol and thymol, which are Vaginal suppositries [4] are dosed drug considered to have antimicrobial activity. The preparations intended for vaginal application. They antibacterial properties of Origanum compactum (O. are usually spherical or conical shape. They are solid compactum) essential oil have been investigated in at room temperature, while they dissolve in vaginal many studies and the obtained results indicate strong secretions at body temperature. antimicrobial activity [5,6]. It is one of the strongest Cocoa butter and similar fatty substances, antibacterial and antifungal oils. It is known for its macrogols or mixtures of gelatin, glycerol and water

ISSN 1840-0426 (P); ISSN 2232-7588 (E) http://tf.untz.ba/technologica-acta 6 M. Ibišević, S. Pilipović, I. Nešić, V. Kerleta, D. Husejnagić, E. Cilović Kozarević, E. Horozić, E. Karić, “Antimicrobial...“, Technologica Acta, vol. 13, no. 2, pp. 5-10, 2020. beneficial effects on respiratory, fungal, urogenital Table 2. Physical characteristics of O. compactum essential oil and other infections. A possible explanation may be Physical characteristics of O. compactum essential oil that carvacrol and thymol participate in the breakdown of the outer membrane of bacteria, Physical state Clear liquid releasing lipopolysaccharides (LPS) and inducing an Colour Golden yellow increase in the permeability of the cytoplasmic Odour Characteristic, phenolic membrane to ATP [6]. Density (20 °C) 0.940 Essential oils can also be unstable, poorly soluble Density (15 °C) 0.943 in water and poorly delivered to target cells. Refractive index (20 °C) 1.505 However, the incorporation of essential oils into Optical rotation (20 ºC) + 0.25 ° liposomes can reduce their irritant effect, while at the Flashpoint 64.4 °C same time prolonging the action of the preparation a itself as well as increasing its effectiveness. (taken from the Quality Specification)

Liposomes are considered highly desirable as drug The chromatogram of O. compactum essential oil is carriers because they may contain hydrophilic, shown in Figure 1, with carvacrol (46.01%, t ,= 82.4 hydrophobic, and amphipathic substances (drugs), ret min), thymol (16.81%, t = 80.7 min), γ-terpinene and are physiologically acceptable also because of ret, (15.61%, t = 24.3 min) and p-cymene (8.08%, t = their similarity to biological membranes and ret, ret, 25.9 min) as the main components. biodegradability [7,8]. These spherical phospholipid vesicles consist of a phospholipid bilayer that surrounds water. The aim of our study was to investigate antimicrobial activity of liposomal and non- liposomal vaginal suppositories, and see if there are any differences in antimicrobial activity.

MATERIALS AND METHODS MATERIAL O. compactum essential oil from Pranarom International (Ghislenghien, Belgique) was used as the active component of the vaginal suppositories Figure 1. Chromatogram of O. compactum essential oil (Table 1, Table 2). It was obtained by hydrodistillation at low pressure and stored at 4 °C. The chemical composition of this essential oil was PREPARATION OF VAGINAL SUPPOSITORIES determined by gas chromatography. Vaginal suppositories were made in two series: Table 1. Basic information about O. compactum essential oil non-liposomal (with O. compactum essential oil) and liposomal (with liposomal dispersion of O. Basic information about O. compactum essential oil compactum) (Table 3). The method of pouring into (taken from the Quality Specification) molds was used. Botanical Origanum compactum Vaginal suppositories were stored in the name refrigerator at a temperature of 4 ºC. After French name Origan compact production, a visual inspection was performed, and Lot number OF31140 the following parameters were recorded: shape, color, Origin PRANAROM - MAROKO odor, presence or absence of fissures. Part of the Flower tops Liposomal dispersion was prepared in the laboratory plant by using high pressure homogenizer (Emulsiflex-C3, Distillation 06/2017 date Avestin, Canada) at 500 bar in five cycles, and pre- Out of date 01/2023 mixing of Phosal 40 IP and O. compactum essential oil with Aqua ad injectabilia [9,10]. This dispersion a (taken from the Quality Specification) contained 9.3% of essential oil.

ISSN 1840-0426 (P); ISSN 2232-7588 (E) http://tf.untz.ba/technologica-acta M. Ibišević, S. Pilipović, I. Nešić, V. Kerleta, D. Husejnagić, E. Cilović Kozarević, E. Horozić, E. Karić, “Antimicrobial...“, Technologica Acta, vol. 13, no. 2, pp. 5-10, 2020. 7

Table 3. Formulations of non-liposomal and liposomal vaginal EXAMINATION OF ANTIMICROBIAL ACTIVITY OF suppositories MANUFACTURED VAGINAL SUPPOSITORIES Components Non-liposomal Liposomal The antimicrobial activity of vaginal vaginal vaginal suppositories suppositories suppositories was examined in the same way as the O. compactum 0.25 g - antimicrobial activity of the essential oil, by the disk essential oil diffusion method [14,15]. Both types of vaginal Liposomal - 2.69 g (0.25 g of suppositories were tested: liposomal and non- dispersion with O. essential oil) liposomal. The prepared vaginal suppositories were compactum dissolved in dimethyl sulfoxide (DMSO). Vaginal Cera alba 2.50 g 2.50 g suppositories were completely dissolved in DMSO, Span 60 0.30 g 0.30 g but the temperature must be taken into account, because at lower temperatures than 37 ºC it hardens. Witepsol 6.95 4.51 g Mueller Hinton (HiMedia, India) agar plates were a (quantities for 5 vaginal suppositories) inoculated with bacterial and fungal suspensions. There were used standard bacterial strains from WEIGHT VARIATION OF VAGINAL SUPPOSITORIES ATCC collection: Staphylococcus aureus (S. aureus) ATCC 25923, Enterococcus faecalis (E. faecalis) The recommended mass of the vaginal ATCC 51299, Escherichia coli (E. coli) ATCC suppositories with respect to the available molds was 25922, Candida albicans (C. albicans) ATCC 10231. 2 g. The prepared vaginal suppositories were Depressions with metal cylinders were made on each evaluated for weight variation according to method plate, in which 100 µL of solution was introduced. of British Pharmacopoeia [11,12]. Twenty vaginal The plates were incubated at 37 °C for 24 hours. suppositories from each series were weighed and the After incubation, the sizes of the inhibition zones, in average values with standard deviation values were millimeters, were measured, in triplicate. Already calculated. measured inhibition zones of essential oil were used

to compare the antimicrobial effect of vaginal DISINTEGRATION TEST suppositories, and a comparison was also made with The disintegration test determines whether the commercial vaginal suppositories (Neo – penotran vaginal suppositories disintegrate at the prescribed forte, Embil Pharmaceuticals CO LTD) . time, under certain experimental conditions in a suitable medium. This test was performed by using a RESULTS AND DISCUSSION magnetic stirrer [13] set at 300 rpm. Phosphate buffer Both types of vaginal suppositories contained pH 4.5 was used as a medium and temperature was 0.05 g of O. compactum essential oil, and the only 37 ± 0.5 ºC, which according to the given conditions difference in the recipe were phospholipids, ie mimics the vaginal environment. The disintegration liposomes in vaginal suppositories with liposomal time was recorded when Witepsol completely melted. dispersion. Both were white color, solid at room According to the recommendations of the British temperature, with a strong characteristic scent of Pharmacopoeia, the disintegration time of vaginal oregano oil (Table 4, Figure 2). suppositories and suppositories should not exceed 60 minutes [12].

Table 4. Physical properties and characterization of prepared vaginal suppositories Properties Non-liposomal vaginal Liposomal vaginal suppositories suppositories Shape Conical Conical Colour White White Odour Strong, characteristic scent of Strong, characteristic scent of oregano oil oregano oil Fissuring - - Weight variation (g) 2.089 ± 0.0447 2.0265 ± 0.0573 Disintegration time (min) 18.16 ± 0.150 31.35 ± 1.35

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deviation should be less than 5%. Non-liposomal vaginal suppositories had a faster disintegration time, due to the simpler formulation, ie the absence of phospholipids. After incubation for 24 hours, inhibition zones (mm) were measured. The obtained results showed a really strong antimicrobial activity of vaginal suppositories (Table 5), because only a small amount of vagitoria solution was introduced on the petri dish (100 µL). Inhibition zones greater than 20 mm indicate the high sensitivity of microorganisms [16,17]. Liposomal vaginal suppositories have almost Figure 2. Vaginal suppositories (own photo) the same activity as non-liposomal vaginal suppositories (Figure 3, Figure 4), but liposomal Both types of vaginal suppositories had an preparations have some other advantages such as average weight about 2g. The weight variation test reduction of irritation potential of essential oil and complies with the regulations of the British prolonged action of the preparation itself. Pharmacopoeia, which states that the standard

Table 5. Antimicrobial activity of prepared vaginal suppositories (non-liposomal and liposomal) Name of the organism Inhibition zones Inhibition zones of Inhibition zones of Inhibition zones of (mm) of Neo – penotran non-liposomal vag. liposomal vag. supp. undiluted O. forte vaginal supp. (mm) (mm) compactum suppositories (100 essential oil (50 µL) µL) E. coli ATCC 25922 35 ± 0.5 24 ± 1,73 16 ± 1.0 10 ± 0.02 E. faecalis ATCC 51299 31 ± 0.57 34,06 ± 2,08 21 ± 0.02 19 ± 1.0 S. aureus ATCC 25923 35 ± 1.15 42,6 ± 2,51 11 ± 1.0 14 ± 1.0 C. albicans ATCC 10231 25 ± 1.0 48 ± 2,0 28 ± 1.0 18 ± 0 a mean ± SD (n=3)

Both types of vaginal suppositories had the largest inhibition zones for C. albicans, and it can be concluded that they have a strong antifungal activity. The inhibition zones were smaller for liposomal vaginal suppositories due to the gradual release of components from liposomes. Many other studies have testified to the positive aspects of encapsulating essential oils [18,19,20]. Neo - penotran forte vaginal suppositories had a larger inhibition zones compared to manufactured vaginal suppositories, but they contain a larger amounts of active ingredients (750 mg of metronidazole and 200 mg of miconazole) than Figure 3. Inhibition zones caused by prepared vaginal supposito- manufactured vaginal suppositories (50 mg of O. ries compactum essential oil).

Figure 4. Antimicrobial activity of non-liposomal and liposomal vaginal suppositories

CONCLUSION [5] S. Bouhdid, S.N. Skali, M. Idaomar, A. Zhiri et al., "Anti- bacterial and antioxidant activities of Origanum compactum Based on the results of this research, it can be essential oil", Afr J Biotechnol; vol. 7, no. 10, pp. 1563- concluded: 1570, 2008.  Origanum compactum essential oil has [6] A. Bouyahya, A. Jamal, F. Edaoudi, A. Et-Touys1,Y. Bakri, N. Dakka, "Origanum compactum Benth: A Review on Phy- shown a strong antimicrobial activity on all tochemistry and Pharmacological Properties", Medicinal tested microorganisms. and Aromatic Plants; vol. 5, pp. 252, 2016.  To the best of our knowledge, vaginal [7] N. Mandić, Ž. Vanić, J. Filipović-Grčić, "Liposomi s suppositories with O. compactum essential kalceinom: odabir optimalne metode priprave", Farm Glas- oil (liposomal and non-liposomal) were nik, vol. 68, pp. 697-708, 2012. prepared and tested for antimicrobial activity [8] S. Franze, F. Selmin, E. Samaritani, P. Minghetti, F. Cilurzo, "Lyophilization of liposomal formulations: Still for the first time. necessary, still challenging", Pharmaceutics, vol. 10, no. 3,  A very small amounts of vagitoria solutions pp. 139, 2018. were tested, but vaginal suppositories also [9] M. Ibišević, V. Tadić, S. Savić, J. Đoković, I. Nešić, "Chap- showed antimicrobial activity on all tested ter 3. Liposome Encapsulation of Origanum compactum Es- strains, especially for C.albicans strain. sential Oil as the Active Ingredient: Assuring the Drug Ef- fect", in The Lamiaceae Family: An Overview. Nova Sci-  Liposomal vaginal suppositories had a ence Publishers. 2020 in press, ISBN: 978-1-53617-078-8. smaller inhibition zones probably due to the [10] M. Ibišević, A. Smajlović, I. Arsić, "Optimization of high slower release of active components, but still pressure homogenization in the production of liposomal dis- have an advantage over non-liposomal persions", Technologica Acta; vol. 12, no.2, pp. 7-10, 2019. vaginal suppositories because they reduce the [11] O. Aremu, Y. Isimi, K. Ekere, O. Agbaje, O. Olayemi, M. irritating potential of the essential oil. Adedokun, M. Emeje, "Comparative evaluation of Irvingia gabonensis seed fat extract and other bases in promethazine hydrochloride suppository formulations", J Res Pharm; vol. ACKNOWLEDGEMENTS 24, no. 2, pp. 240-250, 2020. Authors owe their gratitude to the Faculty of [12] British Pharmacopoeia, British Pharmacopoeia Commission, London, England, 2013. Pharmacy, University of Belgrade, for assistance in [13] T.A. Adegboye, O.A. Itiola, "Physical and release properties this research. of metronidazole suppositories", Trop J Pharm Res; vol. 7, no. 1, pp. 887-896, 2008. REFERENCES [14] S.C. Shivhare, A.R. Umarkar, P.A. Salunke, "Formulation, evaluation and microbial assay of tinidazole vaginal suppos- [1] H.L. Kent, "Epidemiology of vaginitis", Am J Obstet Gyne- itory containing lactic acid bacillus spores", Int J Bioassays; col; vol. 165, pp. 1168-1176, 1991. vol. 3, no.8, pp. 3250-3255, 2014. [2] Ž. Vanić, N. Škalko-Basnet, "Nanopharmaceuticals for im- [15] M. Ibišević, D. Husejnagić, R. Kazanović, I. Arsić, "Anti- proved topical vaginal therapy: Can they deliver?", Eur J bacterial Activity of Origanum Compactum Essential Oil Pharm Sci; vol. 50, pp. 29-41, 2013. Tested on Vaginal and Cervical Clinical Bacterial Strains", [3] S. Bernkop, M. Hornof, American J of Drug Deliv; vol. 1, Acta fac med Naiss; vol. 36, no. 3, pp. 219-228, 2019. pp. 241- 254, 2003. [16] L. Pirvu, C. Hlevca, I. Nicu, C. Bubueanu, “Comparative [4] European Pharmacopoeia; 7th Edition, Strasbourg: Council analytical, antioxidant and antimicrobial activity studies on of Europe, 2010. a series of vegetal extracts prepared from eight plant species

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